original contributions adrenal and circulating …hyper.ahajournals.org/content/20/3/280.full.pdf280...

280

Original Contributions

Adrenal and Circulating Renin-AngiotensinSystem in Stroke-Prone Hypertensive Rats

Shokei Kim, Masatoshi Tokuyama, Masayuki Hosoi, and Kenjiro Yamamoto

The plasma and adrenal renin-angiotensin system in stroke-prone spontaneously hypertensive rats(SHRSP) and Wistar-Kyoto (WKY) rats were examined in animals at 5, 11, 18, and 25 weeks of age.Plasma active renin was significantly increased in 18- and 25-week-old SHRSP with impaired renalfunction, whereas there was no difference in the plasma prorenin level or renal renin content between thetwo strains at all ages examined. Thus, the rate of activation of prorenin seems to be enhanced in thekidney of SHRSP with malignant hypertension. Adrenal renin contents were severalfold higher in SHRSPthan WKY rats at all ages. However, adrenal angiotensin peptides were not increased in SHRSP aged 5and 11 weeks. In 18-week-old SHRSP, adrenal angiotensin II (Ang II) and HI (Ang III) levels werefourfold and 1.8-fold higher, respectively, than in WKY rats, accompanied by 1.5-fold higher plasmaaldosterone. Increased adrenal angiotensin and plasma aldosterone were also found in 25-week-oldSHRSP. Zonal distribution studies indicated that the elevated Ang H and DI in SHRSP were derivedmainly from the capsular tissue (the zona glomerulosa). To examine the contribution of circulatingangiotensin to the adrenal angiotensin content, effects of bilateral nephrectomy on adrenal angiotensinand renin were examined in 18-week-old rats. At 24 hours after nephrectomy, plasma angiotensin,prorenin, and active renin were decreased to almost negligible concentrations. Conversely, in both adrenalcapsular and decapsular tissues of SHRSP and WKY rats, neither angiotensin nor renin was significantlydecreased after nephrectomy. These results suggest that the increase in adrenal capsular Ang II contentsin SHRSP may be partly due to an enhanced local production or Ang II. (Hypertension 1992^20:280-291)

KEY WORDS • renin • renin-angiotensin system • malignant hypertension • angiotensin II •rats, inbred SHR

Multiple lines of evidence1 indicate that variousextrarenal organs, including adrenalgland,2-16 possess an intrinsic renin-angio-

tensin system. Dissociation of hypotensive effects ofangiotensin converting enzyme inhibitor with plasmaangiotensin II (Ang II) levels have been noted inhypertensive patients17 and in rats.18 Either angiotensinconverting enzyme inhibitor or Ang II antagonist cansignificantly alleviate the hypertension in spontaneouslyhypertensive rats (SHR) without high plasma reninlevels.19-20 Thus, the extrarenal renin-angiotensin systemmay play an important role in the pathophysiology ofhypertension.1

Stroke-prone SHR (SHRSP), a substrain of SHRdeveloped by Okamoto et al,21 are a useful model ofhuman malignant hypertension.22-23 The renin-angioten-sin system also participates in the pathophysiology ofmalignant hypertension in SHRSP, as shown by previ-ous reports.24-26 However, previous research done onthe renin-angiotensin system of SHRSP has been lim-ited to the circulating system.22-24-26-29

From the Department of Pharmacology, Osaka City UniversityMedical School, Osaka, Japan.

Supported by a grant from Chichibu Cement and Grant-in-Aidfor Scientific Research 03770110 from the Ministry of Education,Science, and Culture.

Address for correspondence: Shokei Kim, MD, Department ofPharmacology, Osaka City University Medical School, 1-4-54Asahimachi, Abeno, Osaka 545, Japan.

Received December 31,1991; accepted in revised form May 12,1992.

In preliminary experiments, we found that Ang IIcontents in the whole adrenal gland of 25-week-oldSHRSP are higher than those of Wistar-Kyoto (WKY)rats.30 In the present study, we did detailed studies onadrenal and plasma renin-angiotensin systems inSHRSP of various ages. We examined true plasmaprorenin in SHRSP using our recently developedmethods.31-32

MethodsChemicals

Bovine serum albumin (BSA, fraction V) was ob-tained from Seikagaku Kogyo, Tokyo. Trypsin frombovine pancreas (type III, 10,200 Mx-benzoyl-L-arginineethyl ester [BAEE] units/mg protein), soybean trypsininhibitor (type I-S), bovine -y-globulin, and a-methyl-mannoside were from Sigma Chemical Co., St. Louis,Mo. Polyethylene glycol 6,000 was from Fluka, Buchs,Switzerland. Iodine-125-labeled angiotensin I (Ang I)and Ang II and carrier-free sodium ^1 were from NewEngland Nuclear, Boston, Mass. Protein A-Sepharoseand concanavalin A (con A)-Sepharose 4B were fromPharmacia, Uppsala, Sweden. Synthetic angiotensinpeptides were purchased from Peptide Institute, Inc.,Osaka, Japan.

AnimalsSHRSP and Wistar-Kyoto rats were kindly donated

by Dr. K. Okamoto (Kinki University School of Medi-cine, Osaka, Japan) and were maintained by selective

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Kim et al Prorenin and Angiotensin in Hypertension 281

mating at Santen Pharmaceutical Co., Ltd., Osaka,Japan. Animals were fed a standard laboratory chow(CE-1; Clea Japan, Tokyo) and were given tap water adlibitum. Five-, 11-, 18-, and 25-week-old male animals ofboth strains were decapitated, and trunk blood wascollected into prechilled tubes containing ethylenedi-aminetetraacetate disodium (EDTA) (2 mg/ml) andTrasylol (500 kallikrein inhibitor units/ml). The plasmawas separated by centrifugation at 3,000 rpm for 15minutes at 4°C. Bilateral kidneys and adrenal glandswere rapidly removed, weighed, frozen in liquid nitro-gen, and stored together with plasma at -80°C untiluse.

Zonal Distribution of Adrenal Angiotensin and Reninand Effects of Nephrectomy on Adrenal Angiotensinand Renin Contents

In 18-week-old SHRSP and WKY rats, zonal distri-bution of adrenal angiotensin and renin and effects ofnephrectomy on the zonal distribution were examined.Rats were bilaterally nephrectomized by flank incision24 hours before decapitation. Nonnephrectomized and24-hour-nephrectomized rats were decapitated as de-scribed above. After collection of trunk blood, bothadrenal glands were removed, separated into the cap-sular tissue (containing the zona glomerulosa) and thedecapsular tissue (the zona fasciculata/zona reticularisand the medulla), weighed, frozen in liquid nitrogen,and stored at -80°C until use.

Homogenization of Adrenal Gland and KidneyFor measurement of adrenal angiotensin peptides,

whole adrenal glands, capsular tissues, and decapsulartissues were heated at 100°C in boiled distilled water(0.5 ml) for 5 minutes to inactivate proteases, thenhomogenized in 0.05N HC1 solution (total volume, 1ml). The supernatant was obtained by centrifugation at13,000 rpm at 4°C for 1 hour and was assayed forangiotensin peptide contents as described below.

For measurement of active renin, inactive renin,immunoreactive renin, or molecular mass, whole kid-ney, adrenal gland, capsular tissue, and decapsulartissue were homogenized in 50 mM sodium phosphate(pH 7.0) containing 0.1 M NaCl, 2 mM EDTA, and 1mM phenylmethylsulfonyl fluoride (PMSF) and centri-fuged at 13,000 rpm at 4°C for 1 hour to obtain thesupernatant. For kidney, 10% homogenate was pre-pared for all experiments. In the case of adrenal tissue,20% homogenate was prepared for measurement ofactive renin and inactive renin, and 5% homogenate wasprepared for the determination of immunoreactive re-nin and molecular mass and for con A chromatography.

Measurement of Plasma and Adrenal AngiotensinsPlasma and adrenal angiotensin peptides were mea-

sured according to our previously described methods.30

In brief, the sample of plasma (1 ml) and adrenalextracts (1 ml) was applied to a Sep-Pak C18 cartridgecolumn (Waters Associates, Milford, Mass.). The an-giotensins retained by the cartridge were eluted with amixture of methanol/water/trifluoroacetic acid (80/19.9/0.1 vol/vol/vol), dried, dissolved in 10 mM phosphoricacid (pH 3.4), and chromatographed on an ODS-80TMC18 reverse-phase column (4.6 mmx25 cm, Tosoh, Ya-maguchi, Japan). Ang I, II, and HI, Ang (3-8), Ang

(4-8), and Ang (5-8) were completely separated by useof a linear gradient of methanol concentration from30% to 75% in 10 mM phosphoric acid (pH 3.4) over aperiod of 20 minutes at a flow rate of 1.0 ml/min. Eachchromatographic fraction (0.3 ml) was subjected tospecific radioimmunoassay (RIA) of Ang I and II usingrespective antiserum. The anti-Ang II serum usedshowed a 0.02% cross-reactivity with Ang I but a 100%cross-reactivity with Ang III. Thus, Ang III was mea-sured by RIA using anti-Ang II serum. The cross-reactivity of anti-Ang I serum used was 0.05% for eitherAng II or Ang III. The sensitivity of RIA of Ang I andII was 1.5 and 0.3 pg per tube, respectively. Therecovery of Ang I and II, determined by adding theiodine-125-labeled Ang I and II (each 5,000 cpm) to thesample, was 65±4% and 68±5%, respectively, forplasma and 72±3% and 76±3%, respectively, for theadrenal gland.

Measurement of Active ReninActive renin was measured as the rate of Ang I

generation, as described.33 In brief, the samples ofplasma and of renal and adrenal extracts were incu-bated with nephrectomized rat plasma containing anexcess of renin substrate (2 /iM angiotensinogen), andthe Ang I generated was measured by RIA.

Adrenal active renin was regarded as the renin activ-ity inhibitable by specific anti-mature rat renin serum.31

Inhibition of the Angiotensin I-Generating Activity ofAdrenal Extract and Plasma by Anti-Renin Serum

To compare the inhibitory manner of the reninactivity by anti-renin serum31 between adrenal extractand plasma, the samples (100 fil) of adrenal extracts orplasma were incubated with serially diluted anti-reninserum or nonimmunized rabbit serum as control at 4°Cfor 24 hours, and the residual renin activity was mea-sured as described above.

Measurement of Plasma ProreninPlasma prorenin was measured by high-performance

liquid chromatography (HPLC) coupled with trypsinactivation according to our previously described meth-ods.31-32 In brief, the sample of plasma (200-300 /il)from nonnephrectomized and 24-hour-nephrectomizedSHRSP and WKY rats was applied to a G3.000SWcolumn (0.75x60 cm; Tosoh, Yamaguchi, Japan) equil-ibrated with 50 mM sodium phosphate (pH 7.4) con-taining 0.2 M NaCl and 5 mM EDTA. The elution wasperformed at a flow rate of 0.5 ml/min, and fractionswere collected every 30 or 60 seconds. Inactive renin ineach fraction was activated with 0.31 mg/ml trypsin at23°C for 30 minutes, the optimum condition noted inpreliminary experiments. Renin activity after trypsinactivation (total renin) was measured as describedabove. Inactive renin in each fraction was calculated asthe difference between total and active renin. Prelimi-nary experiments showed that the recovery of pure ratrenal active renin34 and purified recombinant rat prore-nin35 from a G3.000SW column was 91±3 (n=6) and89±3% (n=6), respectively.

To determine whether the inactive renin fractionrecovered from the HPLC column was true prorenin,the inactive renin fraction was immunoprecipitated withtwo kinds of anti-prorenin prosegment immunoglobulin



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282 Hypertension Vol 20, No 3 September 1992

G (IgG). The anti-Pro N IgG and anti-Pro C IgG usedrecognize the amino-terminal and the carboxyl-terminalportions of the prosegment of rat prorenin, respectively,as described.31 The immunoprecipitation using proteinA-Sepharose was carried out as described.31 Percentinhibition of the enzymatic activity of inactive reninfraction after trypsin activation by anti-mature rat reninIgG was determined as described.31

Trypsin Treatment of Adrenal and Renal ExtractsTo detect prorenin or inactive renin in the adrenal

gland and kidney, adrenal and renal extracts (50 fil)were incubated with various concentrations (0,0.01,0.1,0.3, or 1 mg/ml) of trypsin at 23°C for 15 or 30 minutes(total volume, 100 /xl), followed by the termination ofthe reaction with the addition of soybean trypsin inhib-itor (2 mg/mg trypsin).31-32 The renin activity aftertrypsin treatment (total renin) was measured as de-scribed above and subtracted from active renin.

Direct Radioimmunoassay of Immunoreactive ReninTo measure immunoreactive renin in plasma and in

renal and adrenal extracts, direct RIA of renin wasdeveloped. To obtain a renin tracer, pure rat renalrenin34 was labeled with ^ 1 by the chloramine T methodand purified by gel permeation HPLC on G3,000SW asdescribed.36 The titer of anti-renin serum used wasx 1,000, and this antiserum was specific for rat renin, asshown by no cross-reactivity to mouse submaxillaryrenin, human renin, and rat cathepsin D and E.31 TheRIA buffer was 50 mM sodium phosphate (pH 7.4)containing 0.15 M NaCl, 3 mM EDTA, 1 mM PMSF,and 0.5% BSA; this was used to dilute all reagents. Theassay incubation mixture consisted of 100 ul standardpure rat renal renin (390 pg to 100 ng) or serially dilutedsample (plasma, renal and adrenal extracts, and chro-matographic fractions of plasma), 100 /xl anti-reninserum (diluted to 1:1,000), and 100 u\ iodine-125-labeled renin and 700 fi\ RIA buffer (total volume, 1ml). After incubation at 4°C for 48 hours, the immuno-complex was separated from the unbound fraction byaddition of 0.1% bovine y-globuUn and 12.5% polyeth-ylene glycol 6,000, followed by centrifugation at 3,000rpm at 4°C for 20 minutes. The radioactivity in theprecipitate was counted with a gamma-counter (Pack-ard model 500C, Rockville, Md.). The lower limit of theassay was 390 pg per tube.

Molecular Mass of Adrenal ReninTo estimate the molecular mass of adrenal renin,

adrenal extracts (300-500 /tl) were subjected to HPLCon G3.000SW as described.36

Concanavalin A Chromatography of Adrenal ReninTo examine whether adrenal renin is glycosylated,

adrenal extracts pooled from 25-week-old SHRSP orWKY rats in 50 mM Tris-HCl (pH 7.2) containing 0.2 MNaCl, 2 mM PMSF, and 10 mM CaCl2 (total volume, 0.5ml) were applied to a con A-Sepharose column (0.5x5cm; bed volume, 1 ml) equilibrated with 50 mM Tris-HCl buffer (pH 7.2) containing 0.2 M NaCl and 1 mMPMSF as previously described.33 After washing with 8ml of the equilibration buffer, the column was elutedwith the equilibration buffer containing 0.2 M a-meth-ylmannoside. The flow rate was 12 ml/hr. Fractions of 1

ml were collected into plastic tubes containing 50 u\equilibration buffer/10% BSA.

Measurement of Plasma AngiotensinogenAngiotensinogen was indirectly measured by incubat-

ing plasma samples with an excess of pure rat renalrenin (50 ng/ml)34 as described.32 The generated Ang Iwas measured by RIA.

Determination of Aldosterone, Blood Urea Nitrogen,and Plasma Creatinine

Plasma aldosterone concentration was determined byRIA using kits (Sorin Biomedica S.p.A., Italy). Bloodurea nitrogen (BUN) and plasma creatinine were mea-sured with kits (Wako Chemicals, Osaka, Japan).

1 8

Ioc

I

' i68K 45X 26K 125K

1 1 , 1 1Inactive renin

Active renin

"20 30 40Retention time (min)

50

BE 12

I

" I68K 45K 25K 1Z5K

1 1 1 1"c—o Inactive renin

>—• Active renin

*•"••* Angioteruinoflen

7 /20 30 40Retention time (min)

800 =

600

400

200 o.2

I50

FIGURE 1. Line graphs show gel permeation high-perfor-mance liquid chromatography (HPLC) profile of plasma fromnonnephrectomized and 24-how-nephrectomized 18-week-old stroke-prone spontaneously hypertensive rats (SHRSP).Panel A: HPLC separation of nonnephrectomized SHRSPplasma resulted in a single peak of inactive renin and activerenin with molecular mass of 48 and 40 kd, respectively. PanelB: Molecular mass of inactive renin from 24-how-nephrec-tomized rats (65 kd) was significantly different from that ofinactive renin from nonnephrectomized rats (48 kd) and ofangiotensinogen (59 kd). Active renin was not detectedthroughout HPLC. Retention time of protein standards includ-ing bovine serum albumin (68 kd), ovalbumin (45 kd),a-chymotrypsinogen (25 kd), and cytochrome C (12.5 kd) isindicated by arrows. Vo, void volume; Ang, angiotensin.



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TABLE 1. Molecular

Molecular mass andimmunoreactivity

Mass and Immunoreactivity of PlasmaIntact

WKY

Inactive Renin

SHRSP

Recovered From HPLC on G3,00OSWNephrectomized

WKY SHRSP

Molecular mass (kd) (n=6)%Immunoreactivity

Anti-Pro N IgG (n=4)Anti-Pro C IgG (n=4)Anti-Pro N IgGcombined with Anti-ProC IgG (n=3)

%Inhibition of the activityby anti-renin IgG (n=4)

47.6±0.4

65±390±3

91±2

92±3

47.8±0J

68±292±3

88±2

91±4

65.2±0.7

2±2

3±2

4±2

643±0.4

2±13±2

3±2

Anti-Pro N IgG and anti-Pro C IgG recognize the amino-terminal and the carboxyl-terminal portions of theprosegment of prorenin, respectively.31 Anti-renin IgG recognizes the portion of mature renin.31 HPLC, high-performance liquid chromatography; WKY, Wistar-Kyoto rats; SHRSP, stroke-prone spontaneously hypertensive rats;IgG, immunoglobulin G.

Measurement of Blood PressureSystolic blood pressure was measured by the tail-cuff

method with a sphygmomanometer (model PS-100,Riken Kaihatsu, Tokyo). Each value is the average ofthree consistent readings.

Statistical MethodResults are expressed as mean±SEM. Statistical

analysis was performed by use of unpaired Student's ftest. Values ofp<0.05 were considered to be statisticallysignificant. Correlation coefficients were calculated bythe method of least squares.

ResultsMethod for Measurement of True Plasma Prorenin

As shown in Figure 1A, the HPLC profile of plasmafrom 18-week-old SHRSP showed a single peak ofinactive renin (trypsin-activatable renin) with a molec-ular mass of 48 kd, a value higher than that of activerenin (40 kd). As shown in Table 1, this inactive reninfraction recovered from HPLC was immunoprecipitatedwith anti-Pro C IgG recognizing the carboxyl-terminalportion of the prosegment by 92±3% (n=4), and the

enzymatic activity of this inactive renin after trypsintreatment was inhibited by specific IgG against maturerat renin (anti-renin IgG) by 91±4% (n=4). Thus, theinactive renin fraction recovered from HPLC was trueprorenin, and HPLC separation of plasma resulted inthe disappearance of inactive renin not caused by eitherprorenin or renin (the trypsin-induced artifact). Therewas no difference in molecular mass and the immuno-reactivity to specific antibody against the prorenin pro-segment or mature renin between the HPLC fraction ofinactive renin from SHRSP and WKY rats at the age of5, 11, 18, and 25 weeks. These observations on SHRSPand WKY rats are consistent with our previous data onWistar rats31-32 that HPLC separation of plasma coupledwith trypsin treatment allows for the measurement oftrue prorenin.

On the other hand, inactive renin from 24-hour-nephrectomized SHRSP and WKY rats had a molecularmass of about 65 kd and was immunoreactive to specificantibody against the prorenin prosegment and maturerenin to a negligible degree (Figure IB and Table 1).Thus, this inactive renin is neither prorenin nor renin.These observations are in good agreement with ourfindings on nephrectomized Wistar rats.31-32

TABLE 2. Body Weight, Blood Pressure, Hematocrit, and Plasma Urea Nitrogen and Creatinine in WKY Ratsand SHRSP

Animals

5 weeks oldWKY (n=8)SHRSP (n=8)

11 weeks oldWKY (n =5)SHRSP (n=5)


25 weeks oldWKY(/i=7)SHRSP (n-7)

Body weight(g)

108±293±3'

282±5234±7*

377±6329±7*

391 ±5320±4*

BP(mm Hg)

107±4113±2

121±3201±8*

135±5249±9*

130±2254±4'

Hct(%)

45.2±0.544.5 ±0.5

44.6±0J44.0±0.8

46J±0.846.6±0.4

44.4±0.743.7±0.4

BUN(mg/dl)

12.0±0.610.3±0.7

11.4±0.711.6±0.6

14.6±1.419.4±0.7*

11.8±0.532.5±3.2»

Plasma creatinine(mg/dl)

0.47±0.010.46±0.01

0.47±0.010.46±0.01

0.61+0.01

0.68±0.03t

0.61 ±0.021.01 ±0.06*

WKY, Wistar-Kyoto rats; SHRSP, stroke-prone spontaneously hypertensive rats; BP, systolic blood pressure; Hct,hematocrit; BUN, blood urea nitrogen.

*p<0.01, tp<0.05 compared with age-matched WKY rats.



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TABLE 3. Plasma Concentrations of Angiotensinogen, Active Renin, Prorenin, Angiotensln, and Aldosterone in WKY Rats and SHRSP

Animals

5 weeks old

WKY (n=8)

SHRSP (TI=8)

11 weeks old

WKY (n=5)

SHRSP (n=5)

18 weeks old

WKY (n=6)

SHRSP (TI=6)

25 weeks old

WKY (TI=7)

SHRSP (7i=7)

Angiotensinogen(ng Ang I • ml"1)

691 ±42597±23*

l,032±63l,112±30

l,010±549O8±59

946±71878±30

Active renin (ngAng I • hi-1 • ml"1)

23.1±23193±2.8

24.8 ±2.622.6±3.2

23.5 ±1.4

42.6±4.4t

14.9±1.2

96.7±13.8t

Prorenin (ngAng I • hr"1 • ml"1)

70.5 ±3.7

40.2±2.2t

30.4±2.229.8±3.1

29.4±1.031.9±4.8

33.9±3.636.5±4.5

Ang I(pg/ml)

129±9133±14

120±4114±8

118±9

201 ±7t

111±15

499±78t

Ang II(pg/ml)

16±215±1

19±116±1

17±1

32±4t

17±290±17f

AnglE(pg/ml)

3.5±0J2.8±0.2

3.2±0.22.9±0.1

2.9±0J4.5 ±0.4'

2.8±0J

13.8±1.5t

Aldosterone(pg/ml)

447±42467±39

408±17411±17

395 ±23

585±25t

324 ±22l,324±156t

WKY, Wistar-Kyoto rats; SHRSP, stroke-prone spontaneously hypertensive rats; Ang, angiotensin.*p<0.05, t/x0.01 compared with age-matched WKY rats.

Plasma Renin-Angiotensin-Aldostemne System inSHRSP and WKY Rats

As shown in Table 2, SHRSP had a normal bloodpressure at age 5 weeks, an elevated blood pressure atage 11 weeks, and severe established hypertension at 18and 25 weeks of age. Both BUN and plasma creatinine of18-week-old SHRSP were significantly higher than thoseof age-matched WKY rats, and these parameters werefurther increased in 25-week-old SHRSP (Table 2).

As shown in Table 3, at 5 weeks of age, there was nodifference in plasma active renin, Ang I, II, and III, andaldosterone concentrations between SHRSP and WKYrats. However, plasma prorenin and angiotensinogen inSHRSP were lower than those in WKY rats. At 11weeks of age, no difference in these parameters inplasma was found between the two strains of rats. In18-week-old SHRSP, the plasma active renin concen-tration was 1.8-fold higher than in the age-matchedWKY rats and was associated with 1.7-, 1.9-, 1.6-, and1.5-fold higher plasma concentrations of Ang I, II, andIII and aldosterone, respectively, in SHRSP. Thesecomponents in the plasma were further increased in

100

80c

£ 60

< 40

20

0

• WKY0 SHRSP

P<0.05

P<0.05

r I18

P<0.01

i25

Age (weeks)

FIGURE 2. Bar graph shows percent active renin in theplasma of 5-, 11-, 18-, and 25-week-old Wistar-Kyoto (WKY)rats and stroke-prone spontaneously hypertensive rats(SHRSP). % Active renin represents the percentage of activerenin to total renin (active renin and prorenin) in plasma.N.S., not significant.

25-week-old SHRSP. In contrast to the increase inplasma active renin, plasma prorenin levels were notelevated in 18- and 25-week-old SHRSP. Thus, thepercentage of active renin to total renin (active reninplus prorenin) in plasma was significantly higher inSHRSP than WKY rats at the ages of 18 weeks(57.5±5.1% versus 44.3±1.7%; p<0.05) and 25 weeks(70.9±4.0% versus 31.1±2.0%;/><0.01) (Figure 2).

In SHRSP, there was a close correlation of plasmaAng II with creatinine (r=0.859; n=26; p<0.01) andBUN (r=0.769; n=26; /><0.01).

Direct Radioimmunoassay of Renin in Plasma and inRenal and Adrenal Extracts

As shown in Figure 3, the dilution curves for renalextracts from SHRSP and WKY rats were parallel to

in

8X

aS

m» •

SHRSP

0.2 as 100

Rsnin (ng) (

FIGURE 3. Line graph shows standard curve for directradioimmunoassay of renin and dilution curves for plasmaand renal extracts from 25-week-old stroke-prone spontane-ously hypertensive rats (SHRSP) and Wistar-Kyoto (WKY)rats. Ordinate (B/B0xl00) is the percentage of bound radio-activity in the presence and absence of unlabeled renin.Abscissa shows the amount of pure renin (A) and the samplevolume of renal extracts from 25-week-old SHRSP (•) andWKY rats (a) (1.5, 3, and 6 yd) and plasma from 25-week-old SHRSP ( • ) and WKY rats (o) (115, 25, 50, and 100 yd),which are plotted on a logarithmic scale.



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TABLE 4. Renal Active Renin and Immunoreacttve Renln Contents and Specific Activity

Animals5 weeks old

WKY (n =8)SHRSP (n =8)


18 weeks oldWKY (n=6)SHRSP («=6)

25 weeks oldWKY(n=7)SHRSP (n=7)

Active renin(jig Angl-hr-'-g"1)

l,264±901,192±99

l,066±511,033 ±68

825±46804±87

781 ±71805±84

Immunoreactive renin(/tg renin/g)

14J±1.413.3±1.2

11.6±0.612.2±1.0

9.8 ±0.710.0±0.9

9.6±1.19.7±1.1

Specific activity*(/ig Ang I • hr"1 • /ig renin"1)

89.1 ±3.490.5 ±3.6

92.5±6.685J±5.2

85.6±5.181.6±6J

83.1 ±4.284.6 ±6.1

Ang, angiotensin; WKY, Wistar-Kyoto rats; SHRSP, stroke-prone spontaneously hypertensive rats.•Specific activity, renin activity divided by immunoreactive renin content

the standard curve for the RIA, thereby indicating theimmunologica] identity between renin in renal extractsand pure renal renin. Thus, the amount of renin in renalextracts could be directly measured (Table 4).

On the other hand, the dilution curves for plasmafrom SHRSP and WKY rats were not parallel to thestandard curve, findings consistent with our previousdata obtained for Wistar rat plasma.31 Although thereason for this is unknown, it is possible that multiplerenin forms with different antigenicity or substance (orsubstances) interfering with binding of antibody to reninmay exist in rat plasma. Thus, this RIA system did notallow us to accurately quantify the amount of immuno-reactive renin in plasma. However, all the plasmasamples from 25-week-old SHRSP (n=7) caused agreater displacement of iodine-125-labeled renin fromthe antiserum than those from WKY rats (n=7),thereby supporting the notion that plasma from 25-week-old SHRSP contains a larger amount of renin thanthat from WKY rats. When chromatographic fractionsof active renin or prorenin obtained by HPLC of plasmawere subjected to direct RIA, immunoreactive reninwas not detected.

Our RIA system was also applied to the directmeasurement of renin in adrenal extracts. In the case ofadrenal extracts (19-63 ng Ang I • hr~' • ml"1) from 5-and 25-week-old WKY rats and 5-week-old SHRSP, noimmunoreactive renin was detected (n=4 for eachgroup). Only in the case of adrenal extracts from25-week-old SHRSP (n=2), whose renin activity was381 and 409 ng Ang I • hr"' • ml"1, respectively, couldimmunoreactive renin be detected. Their immunoreac-tive renin concentrations were 5.21 and 5.33 ng/ml,respectively. The specific activity (the renin activitydivided by immunoreactive renin) was 73.3 and 76.7 figAng I • hr"1 • fig'1 renin, respectively, similar to that ofrenal renin (Table 4).

Renal Active Renin and Immunoreactive Renin inSHRSP and WKY Rats

As shown in Table 4, there was no significant differ-ence in renal active renin and immunoreactive renincontents between SHRSP and WKY rats at all agesexamined. The specific activity (the renin activity di-

vided by immunoreactive renin) was similar between thetwo strains and among all ages examined. These resultsindicate that the decrease in renal renin activity withage can be explained by the decreased amount of renalrenin rather than the appearance of renin isoenzymewith lower enzymatic activity.

Trypsin treatment of renal extracts from 5-, 11-, 18-,and 25-week-old SHRSP and WKY rats (n=4 for eachgroup) did not cause the increase in the renin activity,thereby not allowing for the detection of prorenin orinactive renin in renal tissues.

Renin and Angiotensin Contents in Whole AdrenalTissues of SHRSP and WKY Rats

As shown in Figure 4, in SHRSP at all ages examined,adrenal active renin contents were severalfold higherthan those in WKY rats. Conversely, at age 5 and 11weeks, there was no significant difference in adrenalAng I, II, and III contents between SHRSP and WKYrats, findings indicating a dissociation between adrenalrenin and angiotensin peptide contents. Ang II contentin 18-week-old SHRSP was fourfold higher than inWKY rats (9,494 ±2,568 versus 2,365 ±107 pg/g tissue;p<0.05), in association with a 1.8-fold higher concen-tration of adrenal Ang HI (806±105 versus 443±23 pg/gtissue; p<0.01), whereas the Ang I content was notincreased in 18-week-old SHRSP (446±78 versus404±35 pg/g tissue; p>0.05). At 25 weeks of age,adrenal Ang I, II, and III in SHRSP were 2.3, 4.3, and1.8-fold higher, respectively, than in WKY rats.

The renin activity of adrenal extracts from 5-, 11-,18-, and 25-week-old SHRSP and WKY rats (each,/i=2) was not increased by trypsin treatment. Thus, asin the case of the kidney, neither prorenin nor inactiverenin could be detected in the adrenal gland.

Inhibition of the Angiotensin I-Generating Activity ofAdrenal Extract by Anti-renin Serum

As shown in Figure 5, the pattern of inhibition of theadrenal renin activity by anti-renin serum was similarbetween 25-week-old WKY rats and SHRSP. The max-imum percent inhibition of the activity by anti-reninserum was 98% (the mean of two experiments) for theplasma of both 25-week-old WKY rats and SHRSP and



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89% and 90% (each, the mean of two experiments) forthe adrenal extract of 25-week-old WKY rats andSHRSP, respectively.

Molecular Mass and Affinity for Concanavalin A ofAdrenal Active Renin

The molecular mass of adrenal active renin was 40± 1kd (/i=3), 39±2 kd (n=3), 41 kd (the mean of twoexperiments), and 40 kd (the mean of two experiments)for 25-week-old WKY rats, 25-week-old SHRSP,5-week-old WKY rats, and 5-week-old SHRSP, respec-tively (Figure 6A). The total recovery of adrenal activerenin from the column was more than 85% for allexperiments.

Con A chromatography showed that 84% and 86% ofadrenal active renin from 25-week-old SHRSP andWKY rats, respectively, were retained by a con Acolumn and eluted with 0.2 M a-methylmannoside(Figure 6B). The total recovery of adrenal active reninfrom the column was 73% and 70% for SHRSP andWKY rats, respectively.

M . IAngn T

• P<0.01

- A J Jri n rl J

11 18Age (week*)

FIGURE 4. Bar graphs show renin and angiotensin I, II, andIII contents in the adrenal gland of Wistar-Kyoto (WKY) ratsand stroke-pmne spontaneously hypertensive rats (SHRSP) atthe age of 5, 11, 18, and 25 weeks. Ang, angiotensin; N.S., notsignificant.

Effects of Nephrectomy on PlasmaRenin-Angiotensin-Aldosterone System

To examine whether the elevated adrenal angiotensincontent in SHRSP is a result of the increased uptake ofcirculating angiotensin, the adrenal capsular and decap-sular angiotensin and renin contents were comparedbetween 18-week-old SHRSP and WKY rats at 24 hoursafter nephrectomy. As shown in Figure 7, in bothSHRSP and WKY rats, bilateral nephrectomy remark-ably decreased plasma active renin, prorenin, and AngI, II, and III, yet increased plasma aldosterone. Therewas no difference in plasma active renin (1.22±0.32versus 0.52+0.03 ng Ang I • hr"1 • ml"1), prorenin (notdetected in both groups), Ang I (21.4±3.4 versus14.8±1.7 pg/ml), Ang II (5.4± 1.1 versus 3.2+0.6 pg/ml),and Ang III (0.42±0.12 versus 0.33±0.14 pg/ml) be-tween 24-hour-nephrectomized 18-week-old SHRSPand WKY rats. On the other hand, plasma aldosteroneconcentrations remained higher in SHRSP comparedwith WKY rats after nephrectomy (1,027 ±102 versus719±30 pg/ml; p<0.05).

Effects of Nephrectomy on Adrenal Capsular andDecapsular Angiotensin and Renin Contents

In contrast to a dramatic decrease in plasma Ang I, II,and III and renin 24 hours after nephrectomy, in bothSHRSP and WKY rats, these parameters in the adrenalcapsular or decapsular tissues were not significantlydecreased 24 hours after nephrectomy (Figures 8 and9). In the capsular tissue, active renin and Ang II and IIIcontents (7,180±439 ng Ang I - h r ' - g " 1 , 7,069+729pg/g, and 958 ±95 pg/g, respectively) in nephrectomizedSHRSP were 4.9-, 2.3-, and 1.9-fold higher, respec-tively, than in nephrectomized WKY rats (Figure 8),whereas no difference in Ang I content was foundbetween the two strains of nephrectomized rats(503±56 versus 482±81 pg/g). In the decapsular tissue,renin content (341+29 ng Ang I • hr"1 • g"1) in nephrec-tomized SHRSP was 2.3-fold higher than that in ne-

100

25

Adrenal GlandA WKY

A SHRSP

X10 130 X10001100 X300

Antiserum Dilution

FIGURE 5. Line graph shows percent inhibition of theangiotensin I-generating activity of adrenal extracts andplasma from 25-week-old stroke-prone spontaneously hyper-tensive rats (SHRSP) and Wistar-Kyoto (WKY) rats byanti-renin serum. Dilutions of the antiserum are plotted on alogarithmic scale on the abscissa. Percentage of inhibition ofthe enzymatic activity is shown on the ordinate. Data show themean of two experiments.



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68K45K 2SKI I 1

30 40

Retention time (min)

50

B

I1

&

0.2 M a-methyimannoskto

I

5 10 15

Fraction Number

FIGURE 6. Line graphs show gel permeation high-perfor-mance liquid chromatography on G3,000SW (panel A) andconcanavalin A chromatography (panel B) of adrenal activerenin from 25-week-old stroke-prone spontaneously hyperten-sive rats. Retention time of protein standards is indicated byarrows. Ang, angiotensin.

phrectomized WKY rats, whereas there was no differ-ence in Ang I, II, and III contents between the twogroups (Figure 9) (241 ±49 versus 228 ±35 pg/g,

±266 versus 1,081 ±87 pg/g, and 470±109 versus366±102 pg/g, respectively).

Relation Between Plasma and Adrenal Angiotensin IIConcentrations in SHRSP

Combined data from all SHRSP examined show thatcorrelation between plasma Ang II and adrenal Ang IIconcentrations was weak (r=0.26; n=26; p<0.01).

Relation Between Renin and Angiotensin IIConcentrations in the Plasma and in the AdrenalGland of SHRSP

Figure 10 shows the relation between renin and AngII in plasma and in adrenal gland from all SHRSPexamined. There was a close correlation between reninand Ang II in the plasma (r=0.944; n=26;p<0.01). Onthe other hand, the correlation between renin and AngII in the adrenal gland was weak (r=0.369; «=26;

DiscussionThe concentration and molecular weight of plasma

active renin in SHRSP have been sufficiently examinedby various groups of investigators.22"24-26"29-37 Plasmaactive renin concentrations in SHRSP are increasedwith the development of malignant hypertension. Shi-bota et al23 noted that the increased plasma active reninin SHRSP is associated with an increase in urinaryprotein and renal vascular lesions. Volpe et aJ37 specu-lated that the rise in plasma active renin of SHRSP mayin part contribute to end-organ damage and stroke.However, the plasma Ang II concentrations remained tobe determined. In the present study, we found thatplasma Ang II levels as weh1 as active renin wereincreased in SHRSP with renal dysfunction. Further-more, a close correlation between plasma Ang II andcreatinine or BUN was seen in SHRSP. These observa-tions support the previous assumption23-37 that the cir-culating renin-angiotensin system may contribute to the

Active renin Prorenin

N.S.

Ang I

N> NX

AngH

iIJKLS.

NX

Anglll Aldosterone

.N.S.

NX NX

0—0 WKY •—•SHRSP

FIGURE 7. Line graphs show effects of nephrectomy on plasma active renin, prorenin, angiotensin, and aldosterone levels of18-week-old Wistar-Kyoto (WKY) rats and stroke-prone spontaneously hypertensive rats (SHRSP). Ang, angiotensin; C, nonnephrec-tomized rats; NX, 24-hour-bilateralfy nephrectomized rats. *p<0.05, **p<0.01 compared with WKY rats; N.S., not significant.



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10

I 8I 6

I 4

Bf 2

0

10

8

I 6I 4

2

0

Renin

**

NX

AngD

NX

o—OWKY

1000

1 8 0 °i 600

"I 400200

0

AngI

N.S.»MS.

NX

Anglll

NX

FIGURE 8. Line graphs show effects ofne-phrectomy on adrenal capsular renin andangiotensin contents in 18-week-old Wistar-Kyoto (WKY) rats and stroke-prone sponta-neously hypertensive rats (SHRSP). C, non-nephrectomized rats; NX, 24-how-bilateraltynephrectomized rats; Ang, angiotensin.•*p<0.01 compared with WKY rats. N.S.,not significant.

•SHRSP

exacerbation of malignant hypertension, although therise in plasma active renin seems to be the consequencerather than the cause of renal injury.

In various animals as well as humans, trypsin treat-ment of plasma causes a significant increase in the reninactivity (the Ang I-generating activity).38 This trypsin-induced renin activity is generally called inactive re-nin.38 Using immunological techniques and HPLC, weobtained evidence that trypsin treatment of unfraction-ated rat plasma leads not only to the activation ofprorenin, the biosynthetic precursor of renin, but also tothe occurrence of a significant amount of Ang I-gener-ating activity not related to prorenin or renin (thetrypsin-induced artifact).31-32 Thus, trypsin treatment ofcrude rat plasma does not allow for measurement oftrue prorenin, although the nature of the trypsin-

induced artifact is not fully understood. We developedthe new method for measurement of true rat plasmaprorenin, using HPLC separation of rat plasma onG3,0OOSW before trypsin activation.31-32

In the present study, we first examined true plasmaprorenin in genetically hypertensive rats. At all ages,including the malignant phase, plasma prorenin fromSHRSP was similar to that seen in WKY rats withrespect to the molecular mass and immunoreactivity,thereby differing from the finding obtained for activerenin that plasma active renin from SHRSP in themalignant phase has a significantly larger molecularmass than that from WKY rats.28^29 It should be notedthat despite the progressive increase in plasma activerenin in SHRSP, there was no elevation of plasmaprorenin concentration in SHRSP. In plasma of 18- and

500

| 4 0 0

Jr 300|> 200

ff 100

0

2.5

I 1.5I 1.0

0.5

0

Renin

AngH

NX

KS.

NX

o—OWKY

500

400

, 300

1 200

100

0

1000

600

! 400

200

0

•SHRSP

AngI

N.S.

NX

Anglll

NX

FIGURE 9. Line graphs show effects ofnephrectomy on adrenal decapsular reninand angiotensin contents in 18-week-oldWistar-Kyoto (WKY) rats and stroke-pronespontaneously hypertensive rats (SHRSP). C,nonnephrectomized rats; NX, 24-hour-bilat-erally nephrectomized rats; Ang, angiotensin,*p<0.05 compared with WKY rats. N.S., notsignificant.



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200

I 100

Plasma

„ 20

Adrenal Gland

p«un

0 40 80 120 160

Ptaum rerti (ng Ang I/h/iri)

1000 2000 3000

«•> ("g *"g !/"/(tittw)

FIGURE 10. Scatterplots show relations between renin andangiotensin II levels in plasma (left panel) and adrenal gland(right panel) o/ 5-, 11-, 18-, and 25-week-old stroke-pronespontaneously hypertensive rats. Ang, angiotensin.

25-week-old SHRSP with malignant hypertension, ac-tive renin was the main form, in contrast to prorenin asthe main form in WKY rats. It is possible that theincreased ratio of active renin in plasma may be causedby the in vivo activation of prorenin in the bloodcirculation of SHRSP rather than by an increased renalrelease of active renin. However, we have found thatexogenously administered recombinant prorenin doesnot cause an increase in either blood pressure or plasmaactive renin level in SHRSP39 as well as normotensiverats39 or monkeys,40-41 thereby providing confirmatoryevidence that activation of prorenin does not occur inthe circulation. Thus, the above possibility can beexcluded. Otherwise, it is possible that the increasedplasma renin activity in SHRSP with malignant hyper-tension might be a result of the appearance of reninisoenzyme with greater enzymatic activity. In the pre-sent study, however, plasma from 25-week-old SHRSPcaused a larger displacement of 125I-renin from theantiserum than that from WKY rats, thereby suggestingthat a larger amount of renin exists in plasma fromtwenty-five-week-old SHRSP than in that from WKYrats. Furthermore, the specific activity of renal renin,which is the major source of plasma renin, was similarbetween SHRSP and WKY rats at all ages. Thus, theelevated plasma renin activity in SHRSP seems to be aresult of the increase in amount of active renin, al-though the appearance of renin with greater enzymaticactivity cannot be completely excluded. All these find-ings, taken together with findings of no increase in renalactive renin and immunoreactive renin contents ofSHRSP, indicate that the rate of activation of proreninand the consequent release of active renin are signifi-cantly accelerated in the kidney of SHRSP in the phaseof malignant hypertension.

In the present study, in contrast to the case of plasma,trypsin treatment of renal and adrenal extracts led to noincrease in the renin activity, findings not allowing forthe measurement of prorenin or inactive renin in thesetissues. Several possible reasons for no detection ofprorenin in these tissues are raised. First, it is possiblethat the amount of prorenin in these tissues is in factvery small, if present at all, since prorenin is known tobe rapidly secreted from the tissue without storage (viaa constitutive pathway)42 and exogenously administeredprorenin is readily converted to mature renin withinboth the adrenal gland and kidney.40 Second, prorenin

in these tissues might be converted to active reninduring the homogenization procedure, although thehomogenization buffer contained protease inhibitors.Third, the method for activation used in the presentstudy might not be optimum, although various concen-trations of trypsin were used and two different incuba-tion times (15 and 30 minutes) were used. The accuratemeasurement of tissue prorenin must await the devel-opment of specific and sensitive direct RIA of prorenin.

Previous investigations24-26 revealed a significant hy-potensive action and prevention of organ damage ofangiotensin converting enzyme inhibitor or Ang IIantagonist in SHRSP in the phase of normal plasmarenin level; hence, the extrarenal renin-angiotensinsystem may be involved in the pathogenesis of malignanthypertension in SHRSP. Adrenalectomy markedly de-creases blood pressure in SHRSP43 as well as SHR44-45

but does not in normotensive WKY rats.45 Adrenalmineralocorticoids are suggested to be responsible forthe abnormal vascular reactivity observed in SHRSP.43

Investigations on comparisons of plasma catecholaminelevels between SHRSP and WKY rats46 showed thatplasma catecholamines are higher in SHRSP than inWKY rats, and the adrenal-medullary activity is en-hanced in SHRSP. These findings, taken together withthe finding that Ang II acts on adrenal zona glomerulosacells and medullary cells to stimulate the biosynthesisand release of aldosterone and catecholamines, respec-tively, suggested that we determine adrenal capsularand decapsular angiotensin and renin contents ofSHRSP. The availability of specific anti-mature ratrenin antibody31 allowed for the measurement of trueadrenal renin. Adrenal Ang I, II, and III could bedifferentially measured by HPLC coupled with specificRIA. Adrenal renin and Ang II contents measured inthe present study were about 10-fold and 100-foldhigher, respectively, than those of plasma, findings ingood agreement with reported data.8-15

SHRSP showed severalfold higher adrenal renin con-tents than WKY rats at all ages examined, a findingsimilar to the observation obtained for SHR.3 Of noteare the observations that despite the rise in adrenalrenin, adrenal Ang I, II, and III were not increased in 5 -and 11-week-old SHRSP. Thus, there is a dissociationbetween adrenal renin and angiotensin contents inSHRSP in the phase of no severe hypertension. On theother hand, in 18- and 25-week-old SHRSP with malig-nant hypertension, both adrenal Ang II and III contentswere higher than those of WKY rats. Zonal distributionstudies (Figures 8 and 9) showed that the increasedadrenal Ang II and HI contents are caused mainly bythe capsular portion (the zona glomerulosa).

In the present study, the physicochemical character-istics of adrenal active renin from SHRSP and WKYrats were examined and compared with each other. Nodifference was found between the two strains of ratswith respect to the molecular mass, immunoreactivity,and affinity for con A. Furthermore, all these propertiesof adrenal active renin are similar to those observed foractive renin from the kidney31-33-35 and plasma.31-33 Thus,the increase in adrenal renin activity in SHRSP may becaused by the increased amount of active renin ratherthan the appearance of renin isoenzyme with higherenzymatic activity. This speculation is supported by theobservation that the specific activity of adrenal renin



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from 25-week-old SHRSP was similar to that of renalrenin.

The regulating mechanism of adrenal angiotensincontent remains to be elucidated. Husain et al8 exam-ined the effects of dehydration on rat adrenal capsularand decapsular angiotensin content and Ang II receptorbinding activity and found that adrenal capsular angio-tensin content of dehydrated rats is determined primar-ily by receptor-mediated uptake of circulating angioten-sin rather than the local Ang II production.Autoradiography after injection of iodine-125-labeledAng II47-4* showed that the labeled peptide is preferen-tially taken up by adrenal glomerulosa cells and medul-lary cells by endocytosis followed by transport into thelysosome, thereby supporting the notion that adrenalangiotensin is at least in part derived from the circula-tion. On the other hand, in vitro studies using adrenalcapsular tissue13'16 or cultured dispersed glomerulosacells14'16 provided evidence that glomerulosa cells cansynthesize and secrete Ang II as well as renin. Kifor etal16 have found that superfused rat adrenal capsules canrelease Ang II to a much higher level than the originallycontained Ang II. Thus, adrenal angiotensin content ispossibly affected by the local production as well asuptake of circulating angiotensin.

The present study revealed a weak correlation be-tween plasma Ang II and adrenal Ang II of SHRSP,thereby indicating that the increase in adrenal angio-tensin content of SHRSP cannot be explained only byan increased uptake of circulating Ang II. To furtherexamine the contribution of circulating angiotensin tothe adrenal angiotensin, we studied effects of bilateralnephrectomy on the adrenal capsular and decapsularangiotensin contents. Nephrectomy dramatically de-creased plasma angiotensin and active renin, in agree-ment with reported data.3 Plasma prorenin was notdetectable in either group of rats 24 hours after ne-phrectomy, as in the case of nephrectomized Wistarrats.31-32 Interestingly, in contrast to plasma angiotensinand renin, in both adrenal capsular and decapsulartissues of SHRSP and WKY rats, angiotensin and reninwere not significantly decreased after nephrectomy. Thecapsular Ang II and III remained higher in SHRSP thanin WKY rats after nephrectomy, although there was nodifference in plasma angiotensin levels between ne-phrectomized SHRSP and WKY rats. Thus, the in-creased adrenal capsular Ang II in SHRSP may result inpart from the enhanced local production of Ang II,which might be responsible for a higher plasma aldoste-rone in nephrectomized SHRSP compared with ne-phrectomized WKY rats. However, further study isneeded to elucidate the regulating mechanism of adre-nal angiotensin contents, because the rate of receptor-mediated internalization and degradation of Ang II mayalso affect adrenal Ang II levels.

It is established that renin is the limiting factor in thecirculating renin-angiotensin system.49 In the presentstudy, the close correlation between active renin andAng II in the plasma of SHRSP (Figure 10) confirmsthat in SHRSP, renin is also the most important deter-minant of the circulating Ang II level. Conversely, thecorrelation between renin and Ang II in the adrenalgland of SHRSP was weak. The dissociation betweenadrenal angiotensin and renin was evident in 5- and11-week-old SHRSP and 24-hour-nephrectomized

SHRSP. In addition, investigations on subcellular frac-tionations of the rat adrenal capsules9 and on localiza-tion of adrenal angiotensin converting enzyme7 andangiotensinogen11 showed that the adrenal contents ofboth angiotensin converting enzyme and angiotensino-gen are much smaller than adrenal renin and are notdetectable in zona glomerulosa cells. Therefore, reninmay not be the limiting factor of the adrenal renin-angiotensin system.

In conclusion, the present study shows that the adre-nal capsular Ang II content is increased in SHRSP withmalignant hypertension. This may result in part fromthe enhanced local production of Ang II in the SHRSP.In addition, the rate of activation of prorenin and theensuing release of active renin in the kidney of SHRSPare accelerated with the development of renalimpairment.

AcknowledgmentsWe thank Mariko Ohara for pertinent comments and Dr.

Kazuaki Shimamoto (Sapporo Medical College) for the gift ofanti-Ang II serum.

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S Kim, M Tokuyama, M Hosoi and K YamamotoAdrenal and circulating renin-angiotensin system in stroke-prone hypertensive rats.

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